Metallothioneins (MTs) are thiol-rich polypeptides, expressed in response to metal ion challenges, which sequester, and thus detoxify, metal ions. These polypeptides are also expressed at high levels after cellular exposure to reactive oxygen species. The primary focus of recent work has been to study MT expression and function in response to acute stresses with less attention to potentially more subtle roles during normal cellular metabolism. A need for these more subtle roles is suggested by the observed differential expression of members of this protein class, each presumably with different affinities for various metal ions (eg. copper versus zinc), under different physiological or developmental conditions. Differing affinities likely result from the positioning and type of amino acid side chains within the metal ion binding sites. While much is known about the structure and amino acid composition of the eukaryotic, class I MTs (types 1 and 2), less is known about the bacterial, class II proteins, since the derived amino acid sequences of only three examples (all from the genus Synechococcus spp.) have been published. We developed PCR primers based on the published sequences and have found that genes for the class II MTs (smt A) are widespread among cyanobacteria. The principal aim of the proposed study is to investigate the structure/function relationships of the class II metallothioneins using molecular biology (site-directed mutagenesis), protein biochemistry (metal binding studies) and physical biochemistry (extended x-ray absorption fine structure) approaches. We hypothesize that residues other than thiols, such as appropriately positioned hydroxyls and carboxylates, are important for metal ion binding, and that the positioning and character of such residues is responsible for the differences in affinity toward metal ions exhibited by these polypeptides. A thorough characterization of the metal binding properties of each of the newly identified MTs, and of the effects generated by positioning differing amino acid functional groups within the metal ion binding sites will aid our basic understanding of the functions of these polypeptides under acute and normal conditions.